Monocyte-derived macrophages are critically involved in the initiation and progression of the atherosclerotic plaque in man as well as in experimental models. Plaque rupture is thought to be the trigger event for acute coronary syndromes in man, and several mouse, models of plaque rupture have been described. Macrophages have been implicated in plaque rupture by releasing de-stabilizing proteases. Macrophage apoptosis is prominent in advanced atherosclerotic lesions, but it is controversial whether macrophage apoptosis is beneficial or detrimental. In this proposal we will examine the mechanisms and consequences of macrophage apopotosis in experimental atherogenesis. We propose that macrophage accumulation and activation in the intima contributes to smooth muscle cell death and thereby promotes plaque rupture. Therefore, we will test the primary hypothesis that macrophage apoptosis reduces intimal lesion progression and ultimately contributes to plaque stabilization. As secondary hypotheses, we also propose that Fasmediated signaling plays an important role in macrophage apoptosis and activation in atherogenesis. In Aim 1, we will identify components of the Fas signaling complex regulating activation versus apoptosis in macrophages in vitro, using isotope-coded affinity tag and tandem affinity purification with mass spectrometry. In Aim 2, we will determine the role of apoptosis proteins in macrophage activation and apoptosis in vitro. We will investigate the effect of the anti-apoptotic proteins, c-FLIP, Bcl-2, dominant-negative FADD, and p35 caspase inhibitor, on macrophage activation or apoptosis in response to Fas ligation or atherogenic stimuli. In Aim 3, we will determine the effect of blockade of macrophage apoptosis on lesion progression and plaque rupture in LDLR-/-and ApoE-/-mice. We will transduce hematopoietic stem cells (HSCs) with a novel retrbviral vector incorporating the macrophage-restricted human CD68 promoter and encoding 1 of the antiapoptotic proteins. We will then determine the effect of transplantation of HSCs transduced with a virus encoding 1 of these anti-apoptotic proteins on early and advanced lesions and plaque rupture in LDLR-/- mice. Positive results with transplantion of transduced HSC in the LDLR-/-model will be extended to lesion initiation/progression and plaque rupture in the ApoE-/-model by generating transgenic ApoE-/- mice overexpressing Bcl-2 selectively in macrophages.